Expansion, immunophenotype, and function of virus-specific CB-derived cytotoxic T lymphocytes. (A) For the generation of virus-specific CTLs from CB (virus-CTLs), a total of 2 × 10⁶ CB mononuclear cells (CBMCs) were activated with autologous Ad5f35CMVpp65-transduced dendritic cells (DCs) in the presence of IL-12, IL-7, and IL-15 followed by weekly stimulations with Ad5f35CMVpp65-transduced LCL (LMP2-CTL) and twice weekly IL-2 feeds. CTL cell numbers (×10⁶) are recorded from weekly cell counts comparing the expansion rates of 9 CB-derived CTL lines. (B) Reactivity of CTL lines (n = 9) with antibodies against the T-cell and natural killer cell surface antigens and memory markers CD3, CD4, CD8, CD16, CD56, TCRαβ, TCRγδ, CD45RA, CD28, CD62L, CD25, and the B-cell surface antigen CD19. Error bars indicate mean + SD. (C) ⁵¹Cr release at 4 hours after coincubation of a representative CTL line with PHA blasts pulsed with CMVpp65 PepMix (CMVpp65 target), or PHA blasts pulsed with CMVIE1 PepMix (CMVIE1 target), or PHA blasts pulsed with Adv-hexon PepMix (Adv-hexon target), nontransduced autologous EBV-LCL (EBV target), or allogeneic EBV-LCL (allogeneic target). The data are the percentage of lysis of targets by a representative CTL line at E/T of 40:1, 20:1, 10:1, and 5:1. (D) Virus-specific activity of all 9 CTL lines after 3 stimulations as determined with the IFN-γ ELISPOT assay in response to direct stimulation with CMVpp65 PepMix (CMVpp65), CMVIE1 PepMix (CMVIE1), Adv-hexon PepMix (Adv hexon), or irradiated autologous EBV-LCL at an E/T of 4:1 (EBV). For each CTL line, mean values of triplicate experiments are reported. SFCs indicate spot-forming cells.